Many diseases (e.g., cancers, hematopoietic disorders, endocrine disorders, and immune disorders) arise from the abnormal expression or activity of a particular gene or group of genes. Similarly, disease can result through expression of a mutant form of protein, as well as from expression of viral genes that have been integrated into the genome of their host. The therapeutic benefits of being able to selectively silence these abnormal or foreign genes are obvious.
A number of therapeutic agents designed to inhibit expression of a target gene have been developed, including antisense ribonucleic acid (RNA) (see, e.g., Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994) 91:4504-4508) and hammerhead-based ribozymes (see, e.g., James, H. A, and 1. Gibson, Blood (1998) 91:371). However, both of these agents have inherent limitations. Antisense approaches, using either single-stranded RNA or DNA, act in a 1:1 stoichiometric relationship and thus have low efficacy (Skorski et al., supra). For example, Jansen et al. report that, in a small percentage of patients, relatively high doses (2 mg/kg body weight per day) of antisense RNA resulted in biologically significant levels (i.e., long-term plasma concentrations above 1 mg/L) of encoded protein (Jansen, B., et al., The Lancet (2000) 356:1728-1733). However, no detectable level of plasma protein was observed at lower dosages (e.g., 0.6 mg). Hammerhead ribozymes, which because of their catalytic activity can degrade a higher number of target molecules, have been used to overcome the stoichiometry problem associated with antisense RNA. However, hammerhead ribozymes require specific nucleotide sequences in the target gene, which are not always present.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer enzyme processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature (2000) 404:293-296). In other words, RNAi involves a catalytic-type reaction whereby new siRNAs are generated through successive cleavage of long dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi.
WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.) and Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200). Despite successes in these organisms, until recently the general perception in the art has been that RNAi cannot be made to work in mammals. It was believed that protocols used for invertebrate and plant systems would not be effective in mammals due to the interferon response, which leads to an overall block to translation and the onset of apoptosis (see, e.g., Wianny, F., et al., Nature Cell Biol. (2000) 2:70-75); Fire, A., Trends Genet. (1999) 15:358-363; and Tuschl, T., et al., Genes Dev. (1999) 13(24):3191-97). At least one group of scientists believed that RNAi could only be made to work in mammals if the PKR response could be neutralized or some way avoided, although no suggestions were given as to how this might be achieved (Fire, Trends Genet. (1999), supra; and Montgomery and Fire, Trends Genet. (1998) 14:255-258). However, WO 00/44895 (Limmer) demonstrated for the first time that dsRNA can induce RNAi in mammalian cells, provided that the dsRNA meets certain structural requirements, including a defined length limitation.
Despite significant advances in the field, there remains a need for an agent that can selectively and efficiently silence a target gene using the cell's own RNAi machinery. More specifically, an agent that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target gene at a low dose, would be highly desirable. Compositions comprising such agents would be useful for treating diseases caused by abnormal expression or activity of a gene.